Diadenosine 5 (prime), 5 (triple prime)-P1, P4-tetraphosphate (A(5')pppp(5')A), an acid soluble nucleotide produced from ATP and aminoacyl adenylates in the back reaction of the first step in protein synthesis, has been recently shown by us to be elevated in malignant tissues. A remarkable increase of up to 100 fold in the concentration of Ap4A in transformed cells, as compared to their normal counterparts, has been demonstrated in tissue culture and live animals. High specific activity radioactive labeling of intracellular acid soluble nucleotides, followed by chromatographic separation techniques have enabled the accurate assay of Ap4A in a variety of cells. Levels of 2-2.5 x 10 to the minus 6th power for Ap4A in human hepatoma epithelial cells grown in vivo in the nude mouse or in tissue culture, versus levels of 3-5 x 10 to the minus 8th power M in mouse or rat liver are commonly observed, while the steady state ATP levels in these tissues are similar at 3 x 10 to the minus 3rd power M. A variety of malignant cells showed elevated levels of Ap4A as compared to "normal" untransformed cells in culture. A remarkable metabolic lability, leading to a decrease in Ap4A levels has been demonstrated upon a variety of nutritional shift-downs, amino acid starvation, or serum deprivation of cells in culture. The proposed project includes studies on the intracellular mode of action of Ap4A as well as investigations as to its possible role in malignant transformation. Planned experiments include the analysis of steady state levels and turnover of Ap4A in cells with different growth characteristics; investigation of the intracellular site of action of Ap4A (nucleus, nucleolus, cytoplasm); in vitro studies of possible allosteric control of various glycolytic enzymes by Ap4A and attempts to affect biochemical events (DNA synthesis, RNA, or protein synthesis) in impermeabilized or permeabilized cells or in isolated nuclei by extracellular Ap4A. Proteins which interact with or utilize high specific activity radioactively labeled Ap4A, in tumor tissues and normal liver homogenates will be isolated and characterized. A reaction leading to cleavage of adenine from Ap4A was discovered to be catalyzed by Py-BHK and not by BHK cells. We propose the isolation and characterization of the protein catalyzing this novel "Ap4Ase" (Ap4A nucleosidase (Text Truncated - Exceeds Capacity)